This proposal is aimed at a reliable, quantitative understanding of the internal dynamics of proteins. Time-resolved fluorescence measurements using single-photon counting pulsed methods will be employed in an investigation of the reorientational mobility of tryptophan residues in proteins with emphasis on the buried Trp 138 of T4 phage lysozyme. Site-directed mutagenesis will be used to modify groups neighboring this residue to determine the effect of steric crowding and hydrogen bonding on the motion of this group. Comparison will be made with the more exposed Trp residues of this protein at positions 126 and 158 using single tryptophan variants in which the other two Trp residues have been replaced by tyrosine. The effect of temperature, pH ionic strength and urea concentration, which are known to influence the thermodynamic stability of this protein, will be investigated to see if these external variables influence internal dynamics. Comparison of this system with other single tryptophan proteins will be made, especially pancreatic phospholipase A2 and ribonuclease Tl. All of these proteins will be investigated under conditions of external quenching by iodide at constant ionic strength and when deuterium exchanged, providing additional information for the analysis of complex fluorescence decays and confirming the validity of the analysis of anisotropy behavior. The use of steady state anisotropy measurements as a constraint in the analysis of time resolved anisotropy measurements will be investigated. The origin of the complex photophysics of tryptophan residues in proteins will be investigated using site directed changes adjacent to the tryptophan residues of T4 lysozyme and determination of the effect of such changes on the fluorescence decay. Experiments on crystalline proteins are proposed where diffraction experiments can set limits on the extent of conformational variation. The photophysics of indole derivatives in solution will be further investigated along lines suggested by our recent observations of relaxation events for specific 3-methylindole/butanol complexes. The behavior of 3- methylindole in non-aqueous solution provides a better model for globular protein environments than tryptophan in water. A program of fundamental spectroscopic experiments on indole derivatives is also proposed using high temporal resolution polarization experiments, oriented samples and matrix isolation cryogenic spectroscopy.